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Roumanian Biotechnological Letters Vol. 8, No.1, 2003, pp 1129-1138 Copyright © 2001 Bucharest University Printed in Romania. All right reserved Center for Research in Enzymology and Biotechnology Roumanian Society of Biological Sciences ORIGINAL PAPERS

Synergistic Effect of Bacterial and Fungal Amylases on Breadmaking

Received for publication, November 10, 2002 Published, December 14, 2002

ENUTA IORGA*, NASTASIA BELC*, ALEXANDRU STANCOV*, L. ENACHE, GH. CAMPEANU** * Institute of Food Bioresources, Dinu Vintila Street No. 6, sector2, 72136, Bucharest, Romania ** Faculty of Biotechnology, Bd. Marasti No. 59, sector 1, 71331, Bucharest, Romania

Abstract For decades enzymes have been used for bread making. Due to the changes in the baking industry and the demand for more varied and natural products, enzymes have gained more and more importance in bread formulations. The study evaluates the synergistic effects of these enzymes when these enzymes are combined with each other. Bread staling is responsible for significant financial losses. A maltogenic - amylase is a true anti-staling enzyme that affects neither volume or crumb structure. Therefore, it is more feasible to use this enzyme in combination with enzymes such as fungal -amylase to ensure the other bread quality parameters such as volume, dough stability, and crumb structure. Concerning the interaction of enzymes substrates in dough and bread is rather complex, using of enzyme combinations should have synergistic effects that are not observed if only one enzyme is used – even at high dosages. The added enzymes (Novamyl with Fungamyl 2500 BG, Novamyl with fungal -amylase, respectively) influence have been established by baking tests. The maltogenic -amylase has the synergistic effect together with two fungal -amylases: 45 ppm of maltogenic together with other enzymes gave a softer crumb during storage than using 75, 100 or 200 ppm of the maltogenic -amylase alone.

Key words: bacterial -amylase, fungal -amylase, synergistic effect, breadmaking, etc.

Introduction Bread is the most common traditional food in the world, with low cost. Today, bread is closely related to biotechnology, the term that relates to these two subjects is enzyme. Using enzyme combinations for breadmaking is not new. Enzymes are used for dough conditioning, for extending shelf life and improving crumb softness and elasticity, and for dough strengthening. The maltogenic -amylase has a unique anti-staling effect. This article reviews the synergistic effects of maltogenic -amylase with traditional enzymes such as fungal -amylase. Therefore, the objectives of this research were to determine: 1) the synergistic effects of Novamyl and Fungamyl 2500 BG on bread quality parameters; 2) the synergistic effects of Novamyl and fungal -amylase from S.C.Enzymes & Derivates Romania S.A. on bread quality parameters; 3) the effects of enzyme combinations on the gases power and retention in dough; 4) the results repeatability. 1129 ENUTA IORGA, NASTASIA BELC, ALEXANDRU STANCOV, L. ENACHE, GH. CAMPEANU Materials and Methods

Commercial flours were obtained from S.C. PLEVNEI S.A. and S.C. BANEASA S.A. The physico-chemical and rheological indicators are given in the (Table 1 and Table 2).

Table 1. Physico-chemical indicators of flours. Qualitative Indicators UM Values F1 F2 Moisture % 14.81 15.31 Ash % d.m. 0.69 0.44 Acidity degrees 2.4 2.4 Wet gluten % 24.3 22.4 Gluten Index 95 99 Gluten deformation mm 3.5 3.0 Falling Number sec. 383 347

Table 2. Rheological indicators of flours. Rheological Indicators UM Values F1 F2 Hydration capacity % 55.9 52.8 Development min. 2.0 1.8 Stability min. 4 3.5 Elasticity uB 130 130 Softening uB 110 90 Power - 40 42

The amilolitics enzymes used were the following: Maltogenic -amylase: Novamyl, Novo Nordisk, Denmark. Fungal -amylases: Fungamyl 2500 BG, Novo Nordisk, Denmark; fungal -amylase, S.C. Enzymes & Derivates Romania S.A. Pakmaya yeast was used from Rompak Ltd., Romania.

Baking The enzymes were added to the baking formula during the mixing stage. Breads were made with commercial bread flour 480 and 650 type. Employing the pup loaf formula with a 90-min. fermentation, straight-dough process (Romanian Standard STAS 90-88 Baking Test). The dough were proofed and then baked. The loaf volume of bread was measured using rapeseed displacement. Physico-chemical indicators of all the breads were determined and the internal and external characteristics evaluated sensorial properties.

Freshness determination The Freshness analysis was performed with an L MIM Penetrometer. The method consists in measurement of the resistance opposed, to an applied force, for 10 seconds, on bread crumb. The measurements of freshness are made at 3, 24, 48, 72 and 96 hours, respectively, the results being compared with the control sample at the same time periods.

106 Roum. Biotechnol. Lett., Vol. 8, No. 1, 1129-1138 (2003) Synergistic effect of bacterial and fungal amylases on breadmaking

Power Gas The straight-dough formula with 250 g of commercial flour, 5.0 g of salt, 5.0 g of yeast and water was used for the power gas test (a strengthened dough is obtained). Bacterial and fungal alpha-amylases were added to some dough were in different quantities to notice the optimal doses which can be used for a better quality of bread. The control dough had no additives. Zymotachigraph Chopin was used for power and retention gas.

Results and Discussions To compare the performance of bacterial and fungal -amylases on bread quality in baking systems have to be optimized technological parameters and the doses of additives.

The Influence of Novamyl and Fungamyl 2500 BG on bread quality

Flour F1 Table 3. Recipes and technological parameters in baking test.

Raw materials Direct method F1 Control P14 P15 P16 P17 Flour, kg 2 2 2 2 2 Water, l 1.12 1.12 1.12 1.12 1.12 Yeast, g 60 60 60 60 60 Salt, g 30 30 30 30 30 Novamyl, g + - 0.09 0.09 0.09 0.09 Fungamyl 2500 BG, g - 0.01 0.02 0.04 0.1 Mixing time, min. 3 3 3 3 3 Fermentation time, min. 90 90 90 90 90 Proofing time, min. 65 65 65 65 65 Baking time, min. 30 30 30 30 30 Baking temperature, °C 230

The following quantities of Novamyl were used: 0.045 g/kg flour, which correspond with 450 units MANU/kg flour and Fungamyl 2500 BG: 0.005; 0.01; 0.02 and 0.05 g/kg flour, respectively, which correspond to 12.5; 25; 50 and 125 units FAU/kg flour, respectively. The bread was packed 3 hours from the baking.

Table 4. Quality indicators of bread after 24, 48, 72 and 96 hours, respectively from the baking. Sample/Parameters Control P14 P15 P16 P17 Weight, g 520 527 534 537 532 529 524 526 525 530 537 526 529 527 536 532 521 516 528 533 Roum. Biotechnol. Lett., Vol. 8, No. 1, 1129-1138 (2003) 107 ENUTA IORGA, NASTASIA BELC, ALEXANDRU STANCOV, L. ENACHE, GH. CAMPEANU

Volume, cmc/100 g 313 359 343 340 334 294 344 342 347 340 302 349 340 338 327 312 365 365 345 336 Height, cm 9.4 11.1 11.1 10.7 10.7 9.8 11.2 10.7 10.3 10.3 10.0 10.7 11.0 11.3 10.8 9.9 10.9 10.7 11.0 11.2 Diameter, cm 15.45 15.45 15.65 15.95 15.35 15.55 15.05 14.9 15.3 15.35 15.55 15.35 15.25 15.2 15.05 15.4 15.2 15.45 15.45 14.9 H/D 0.61 0.72 0.71 0.67 0.70 0.63 0.74 0.72 0.67 0.67 0.64 0.70 0.72 0.74 0.72 0.64 0.72 0.69 0.71 0.74 Porosity (Crumb 82 86 85 84 83 structure), % 82 86 85 82 86 82 85 84 84 85 81 85 82 83 85 Elasticity, % 98 98 98 97 98 97 98 100 97 98 95 98 97 98 98 95 97 98 98 98 Moisture, % 44.19 44.0 44.50 44.21 44.40 43.31 44.4 43.91 44.20 43.80 43.89 42.89 44.1 44.4 44.40 43.90 43.91 42.80 44.01 44.31 Acidity, degrees 1.4 1.2 1.6 1.2 1.0 1.2 1.4 1.4 1.0 1.2 1.2 1.2 1.4 1.2 1.2 1.2 1.6 1.6 1.6 1.4 Bread score 90 97 96 95 90 87 96 96 92 94 84 95 92 91 94 85 96 93 93 94 Freshness 518 596 521 554 634 481 592 574 525 602 536 527 516 474 545 485 513 508 513 557

Influence of Novam yl and Fungam yl 2500 BG Influence of Novam yl and Fungam yl 2500 BG on bread volum e in tim e on bread elasticity in tim e 370 100

360 99 g 0

0 350 1 /

c 98 %

340 , m y c t

i , c e 97 330 i Control Control t m s u a

l P14 320 P14 l o E 96

V P15 310 P15 108 Roum. Biotechnol. Lett., Vol. 8, No. 1, 1129-1138 (2003)P16 P16 95 300 P17 P17 290 94 24 48 72 96 24 48 72 96 Tim e, hours Tim e, hours Synergistic effect of bacterial and fungal amylases on breadmaking

Variation of bread volum e taking into Variation of bread H/D taking into account account the used enzym e doses the used enzym e doses 370 0,74 360 0,72 350 0,7 g

0

0 340 0,68 1 D / / c H

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, e 320 0,64 m u l 310 0,62 o V 300 0,6 Control 450 MANU + 450 MANU + 450 MANU + 450 MANU + Control 450 MANU + 450 MANU + 450 MANU + 450 MANU + 12.5 FAU 25 FAU 50 FAU 125 FAU 12.5 FAU 25 FAU 50 FAU 125 FAU Dose, units/kg flour Dose, units/kg flour

Figure 1. Variation of physico-chemical parameters of bread obtained from baking tests with Novamyl and Fungamyl 2500 BG.

Influence of Novam yl and Fungam yl 2500 BG Influence of Novam yl and Fungam yl 2500 BG on bread freshness in tim e on bread score in tim e 650 98 Control 630 P14 96 610 g P15 0 94 0 590 P16 1 % /

, c 570 P17 92 e m t a c Control

t

, 550 i 90 c i m P14 t

u 530 s l 88 P15 a o l

V 510 E P16 86 490 P17 470 84 450 82 24 48 72 96 24 48 72 96 Tim e, hours Tim e, hours

Figure 2. Variation of freshness and bread score, in time, of bread obtained from baking tests with Novamyl and Fungamyl 2500 BG. By the analysis of the table information and the graphic representations it results that the highest values of the bread quality indices were obtained at P14 sample, with Novamyl addition (constant dose) and Fungamyl 2500 BG (the lowest dose). We should remark that the values obtained for the other samples are also higher than the values of the control sample. By the analysis of the results we can come to the following conclusions: the values of the bread quality indices obtained by the simultaneous action of the two enzymes are higher than those resulted by the individual action of each enzyme preparation; on the other hand, the

Roum. Biotechnol. Lett., Vol. 8, No. 1, 1129-1138 (2003) 109 ENUTA IORGA, NASTASIA BELC, ALEXANDRU STANCOV, L. ENACHE, GH. CAMPEANU optimal utilization dose for each enzyme preparation, in case of simultaneous action, is much lower than in the case of separate utilization.

The Influence of Novamyl and Fungamyl 2500 BG on gas power Experiments for gas power determinations were made.

Table 5. CO2 volumes and retention surface (Flour F1). Parameters Control P14 P15 P16 P17 3 Total CO2 volume, cm 2700 2430 2490 2400 2535 Total surface, cm2 180 162 166 160 169 Retention surface, cm2 36 16 16 15 16 Time, hours 4 4 4 4 4 Temperature, °C 30 28 28 28 29

From the table above one can noticed that total volume of CO2 is not higher than the control sample. Maltogenic -amylase does not affect the dough rheological property because of its low activity at a temperature less than 35°C. The values obtained did not correlate with technological tests, where the best results are for sample P14.

The Influence of Novamyl and Fungamyl 2500 BG, Novamyl and fungal -amylase, respectively, on bread quality

Flour F2 Table 6. Recipes and technological parameters in baking test. Raw materials and Direct method technological regime F2 Contro P12 P13 P14 P15 l Flour, kg 1.5 1.5 1.5 1.5 1.5 Water, l 0.8 0.8 0.8 0.8 0.8 Yeast, g 45 45 45 45 45 Salt, g 22.5 22.5 22.5 22.5 22.5 Novamyl + - 0.0675 - - - Fungamyl 2500 BG, g 0.0075 Novamyl, g + - - 0.0675 0.0675 0.0675 Fungal -amylase, g 0.0015 0.0034 0.0068 Mixing time, min. 3 3 3 3 3 Fermentation time, min. 90 90 90 90 90 Proofing time, min. 65 65 65 65 65 Baking time, min. 30 30 30 30 30 Baking temperature, °C 230 The following quantities of Novamyl were used: 0.045 g/kg flour, which correspond to 450 units MANU/kg flour and Fungamyl 2500 BG: 0.005 g/kg flour, respectively, which correspond to 12.5 units FAU/kg flour, respectively (sample P1); Novamyl 0.045 g/kg flour, which correspond to 450 units MANU/kg flour and fungal -amylase from S.C. Enzymes & Derivates Romania S.A.: 0.001; 0.0023 and 0.0045 g/kg flour, respectively, which correspond to 40; 90 and 180 units SKB/kg flour, respectively. The bread was packed after 3 hours from the baking. 110 Roum. Biotechnol. Lett., Vol. 8, No. 1, 1129-1138 (2003) Synergistic effect of bacterial and fungal amylases on breadmaking

Table 7. Quality indicators of bread after 3, 24, 48 and 72 hours, respectively from the baking. Sample/Parameters Control P12 P13 P14 P15

Weight, g 518 490 521 515 525 495 517 524 517 530 517 513 529 486 489 511 519 489 510 519 Volume, cmc/100 g 326 362 345 353 353 328 350 312 329 328 311 351 327 342 352 333 345 340 353 334 Height, cm 10.2 9.8 10.7 11.0 11.0 10.5 11.2 10.4 10.9 10.0 10.5 11.5 11.1 10.5 10.2 10.6 11.5 10.7 11.0 10.2 Diameter, cm 16 16.1 16.1 15.8 16.1 15.3 15.05 15.0 15.05 15.45 15.45 15.25 15.1 15.5 15.3 15.3 15.55 15.75 15.4 15.6 H/D 0.64 0.61 0.66 0.70 0.68 0.69 0.74 0.69 0.72 0.65 0.68 0.75 0.74 0.68 0.67 0.69 0.74 0.68 0.71 0.65 Porosity (Crumb structure), % 83.5 86.2 85.68 82.56 85.68 83.4 84.8 81.4 84.82 85.3 82.94 87.6 84.45 84.92 85.11 83.79 85.4 83.97 84.73 84.73 Elasticity, % 95 95 98 98 97 98 98 97 98 98 98 100 98 98 97 98 97 98 98 97 Moisture, % 43.8 43.90 43.01 44.19 44.10 43.71 43.90 43.39 44.21 43.99 43.11 43.50 44.70 42.71 43.5 43.00 43.11 42.71 43.09 43.10 Acidity, degrees 1.0 0.8 0.8 0.8 1.0 1.2 1.0 1.0 1.0 1.0 1.0 1.0 0.8 1.2 0.8 1.0 0.8 1.0 1.2 1.0 Bread score 88 93 93 93 95 87 92 86 92 92 85 92 91 93 93 89 92 90 93 91 Freshness, mm 630 640 600 605 615 522 635 563 612 573 513 605 589 592 516 InfluenceInfluence of Novam of Novam yl and yl Fungam and 554Fungam yl 2500 yl BG2500 BG603 InfluenceInfluence532 of Novam of Novam yl and555 yl Fungam and Fungam yl 2500 yl BG2500562 BG FigureVariation 3. Influenceon breadofon bread bread of volum Novamyl volum freshness e in e tim takingand e inFungamyl timinto e 2500 BG, Novamyl and fungal -amylase from S.C. Enzymes 680 Variationon ofbread breadon elasticity bread H/D taking score in tim into in etim account e 100 100 370 account& the Derivates used enzym Romania e doses S.A., respectively,Control on bread physico-chemicalthe used enzym indicators. e dos es Control 370 660 P12 0,72 P12 360 98 g P13 99 P13

g 640 0 360 0 0 0,7 96

350 0 1 P14 P14 / 1

620 % / c 350 98 , % g c P15

P15

94 m e 340 , 0,68 0 t m c 600 y

0 a t c , i

340 t 1 i , D e / c 330 / 97 92 i 0,66c c i Control t

m 580 m H t s u

u m 330 Control s l l a c 320 l 90

P12 a o o 560 l , E 960,64 P12 V V E e 320 310 P13 88 m 540 P13111

u Roum. Biotechnol. Lett., Vol. 8, No. 1, 1129-1138 (2003) l 310 P14 950,62 o 300 520 86 P14 V P15 P15 290300 0,6 500Control 450 MANU + 450 MANU + 450 MANU + 450 MANU + 94 84Control 450 MANU + 450 MANU + 450 MANU + 450 MANU + 0 0 2412.5 FAU 24 4048 SKB 90 SKB4872 180 SKB 72 0 0 2412.5 FAU 244048 SKB 90 SKB7248 180 SKB 72 TimDose, e, hours units/kgTim e, hours flour TimDose, e, hours units/kgTim e, flour hours ENUTA IORGA, NASTASIA BELC, ALEXANDRU STANCOV, L. ENACHE, GH. CAMPEANU

Figure 4. Influence of Novamyl and Fungamyl 2500 BG, Novamyl and fungal -amylase from S.C.Enzymes & Derivates Romania S.A., respectively, on bread freshness and bread score in time.

In the case of flour F2 two possibilities were tested: - the enzymes doses which gave the best results in the case of flour F1, for reproducibility; - the same dose of Novamyl and different doses of fungal -amylase. The conclusions were: - synergetic effect of the two enzymes is the same for the flour F1 and F2; - for the flour F2, the best synergistic effect is for the sample with 450 units MANU/kg flour and 90 units SKB/kg flour. Finally, the values of physico-chemical indicators for the samples with added enzymes are certainly higher than those of the control sample and have a good steadiness in time.

Variation of bread volum e in tim e Variation of bread porosity in tim e 375 90 Flour 1 Flour 2 365 89 Flour 1 Flour 2 355 88 g

0 3h 3

0 345 87 1 % /

24h , 24 c 335 86 y t m i

c 48h s 48

325 85 , o r e

315 72h o 84 72 m P u l 305 83

o 96h 96 V 295 82 285 81 275 80 Control 450 MANU + Control 450 MANU + Control 450 MANU + Control 450 MANU + 12.5 FAU 12.5 FAU 12.5 FAU 12.5 FAU Flour sam ples Flour sam ples

Variation of bread freshness in tim e Variation of bread score in tim e 650 Flour 1 100 Flour 2 Flour 1 Flour 2 630 98 610 96 3 3 m 590 94 m

24 24 , 570 e 92 r s s o e 550 48 c 90 48 n S h 530 72 88 72 s e r 510 86 F 96 96 490 84 470 82 450 80 Control 450 MANU + Control 450 MANU + Control 450 MANU + Control 450 MANU + 12.5 FAU 12.5 FAU 12.5 FAU 12.5 FAU 112 Flour sam ples Roum. Biotechnol. Lett., Vol. 8,Flour No. sam 1, ples1129-1138 (2003) Synergistic effect of bacterial and fungal amylases on breadmaking

Figure 5. Synergistic effect of Novamyl and Fungamyl 2500 BG.

Figure 5 presents the variations of the quality indices for the samples obtained from the two flours with enzyme preparation supplements, in optimal doses experimentally established for the case of their simultaneous action. From the statistical analysis of the data it results that the synergic effect of the utilization of the two types of enzyme preparations, having beneficial effects on the quality sensory indices, as well as on the bread freshness.

Conclusions In the following table it is represented the synergistic effect of the enzymes used on the experiments.

Table 8. Synergistic effect of bacterial and fungal amylases on breadmaking

Name of Activity Flour Optimal dose commercial Falling (to flour) product Number, sec. Novamyl + 10000 MANU/g 383 347 450 450 Fungamyl 2500 BG 2500 FAU/g MANU/kg MANU/kg flour flour 12,5 FAU/kg 12,5 FAU/kg flour flour Novamyl + 10000 MANU/g 347 450 MANU/kg flour Fungal alpha- 40000 SKB/g 90 SKB/kg flour amylase

1. Optimal doses for the flours used in experiments were determined, as we can see in table 8. 2. The quality indices (volume, porosity, elasticity, bread mark, etc.) have improved. The values of bread quality indices resulted by the simultaneous action of the two enzyme preparations are higher than those resulted by the individual action of each of them separately. 3. The sensory characteristics generally improved, fact which reflects the variation of the bread mark. 4. The influence of the combination enzyme, Novamyl and Fungamyl 2500BG on the total CO2 volume produced within the dough was examined. The resulted values are not correlated to the results of the technological samples, when the best results were obtained at P14 sample. higher than those of the control sample and are constant in time. 5. In parallel, the influence of the enzyme combination on the bread freshness was examined. The obtained values are higher than those of the control sample and remain constant in time. 6. The synergic effect of the two enzymes, Novamyl and Fungamyl 2500BG, was ascertained on both flour number 1 and number 2. A good experimental reproduction of the enzyme doses was obtained, as well as of the results, which can constitute a data base, subsequently used at industrial level.

Roum. Biotechnol. Lett., Vol. 8, No. 1, 1129-1138 (2003) 113 ENUTA IORGA, NASTASIA BELC, ALEXANDRU STANCOV, L. ENACHE, GH. CAMPEANU

7. In the case of the second enzyme combination, Novamil and fungi -amylase, at flour number 2, the synergic effect resulted for lower enzyme doses than those established in the case of their individual action.

Acknowledgements The experiments were financed by the RELANSIN PROJECT 966/2001

References 1. Romanian Standard 90-88. Baking Test 2. Zymotachigraphic method.

114 Roum. Biotechnol. Lett., Vol. 8, No. 1, 1129-1138 (2003)

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